Study of the immune system has shown it to be an extremely complex one, requiring countless signals in order to function optimally. See, e.g., Janeway, Quant. Biol., 54:1-4 (1989), Paul, ed., Fundamental Immunology, (4th ed., 1998), both of which are incorporated by reference herein.
An essential feature of an immune response are the interactions between T lymphocytes and antigen presenting cells (“APCs”). Many cohesive molecules that are found on T cells and APCs increase during an immune response. Increased levels of these molecules are believed to be key to the fact that activated APCs are more effective at stimulating antigen specific T cell proliferation, than are resting APCs.
It is thus not surprising that the T cell immune response is a complex process, involving cell interactions, including those between T cells and accessory cells, including APCs, and production of soluble immune mediators. This T cell response is regulated by various T-cell surface receptors, including, but not being limited to, the T-cell receptor complex, as well as other “accessory” surface molecules. Many of these accessory molecules are naturally occurring cell surface differentiation (CD) antigens defined by the reactivity of monoclonal antibodies on the surface of cells.
It is well established that, on CD8TLs, the TCR and CD8 molecules co-participate in the recognition of a complex formed by an antigenic peptide, and a class I major histocompatibility complex, or “MHC” molecule. In humans, these MHC molecules are known as “human leukocyte antigens” or “HLAs.” When “HLA” is used herein, it is to be understood that it is representative of MHCs generally.
Details of the aforementioned interaction are well established. TCRs are dimers, with “α” and “β” chains, and are sometimes referred to as “TCRabs”. These molecules interact with the peptide, and the surrounding grove of the MHC class I molecule, with a kD generally higher than 3 mM, in syngeneic responses. The CD8 molecule, also heterodimeric, and sometimes referred to as CD8αβ, contacts the αβ constant region of the MHC class I molecules, with a kD of approximately 100 mM. See, Arcaro, et al., J. Exp. Med., 194:1485-1495 (2001); Holler, et al., Immunity, 18:225-264 (2003).
When complexes of TCR and CD8 form, the resulting complexes have affinities for the MHC-peptide complexes that are about ten-fold higher than the TCR alone. Holler, supra.
Structurally, CD8αβ molecules are located in lipid rafts, in association with intracellular tyrosine kinase p56lck. Arcaro, et al., supra, Arcaro, et al., J. Immunol., 165:2068-2076 (2000). TCRs are associated with CD3γ, δ, ε and ζ chains, and are not located in rafts, but TCR-CD3 complexes interact closely with CD8-p56lck complexes that are found in rafts, so as to induce signal transduction when binding to antigen. Doucey, et al., Eur. J. Immunol., 31:1561-1570 (2001); Montixi, et al., EMBO J., 17:5334-5348 (1998).
After the initial binding of CD8TLs to target cells, additional TCRs congregate at the contact region, referred to as the “immunological synapse.” Synapses mature to form distinct patterns with TCR molecules located in the center and surrounded by LFA-1 adhesion molecules, which are in turn surrounded by CD45 molecules. Huppa, et al., Nat. Rev. Immunol., 3:973-983 (2003); van der Merwe, et al., Semin. Immunol., 12:5-21 (2000). Further, the microtubule organizing center, or “MTOC” moves to the synapse, directing release of lytic granules into target cells. Grakoui, et al., Science, 285:221-227 (1999); Stinchcombe, et al., Immunity, 15:751-761 (2001). During the process, TCR molecules in the synapse begin internalizing, resulting in a considerable decrease of TCRs on the cell surface; however, newly synthesized and recycled TCRs move to the cell surface, and the initial level of TCRs on cell surfaces is recovered, usually, within 24 hours of antigen presentation. Valitutti, et al., Nature, 375:148-151 (1995).
It is also well known that if CD8TLs are stimulated, e.g., weekly, with their target antigen in the presence of growth factors, they can be kept in culture for several months. It has been observed, however, that their cytolytic activity can diminish or be lost completely.
Demotte, et al., Eur. J. Immunol., 32:1688-1697 (2002), the disclosure of which is incorporated by reference, observed that CD8+ T lymphocytes lose their specific cytolytic activity if, instead of being stimulated weekly with tumor cells, are stimulated with Epstein Barr Virus (“EBV”) transformed B cells which present the relevant antigenic peptide. The loss of effector function is always associated with loss of labeling by HLA-peptide tetramers. While Demotte et al. were not able to identify the reason for this loss of activity, they did eliminate loss of TCR or CD8 expression as reasons.
Galectin-3 is a well known molecule. An exemplary, but by no means exhaustive listing of the literature on this molecule includes Dumic, et al., Biophysica. Acta., 616-635 (2006), Demetriou, et al., Nature, 409:733-739 (2001), both incorporated by reference, which provide details on the structure of galectin-3, as well as a role in forming a lattice, with glycoproteins, leading to restriction in recruitment of TCRs to antigen presentation sites.
Also of interest are U.S. Pat. No. 6,680,306, describing how surgical procedures can be improved by administering a carbohydrate which selectively binds to a galectin, including galectin-3; U.S. Pat. No. 6,890,906, which discusses how the use of complex carbohydrate molecules which bind to galectins, including galectin-3, can control angiogenesis, and U.S. Pat. No. 6,770,622 which describes a role for galectin-3 in tumor metastasis, and describes a truncated galectin-3 molecule useful in treating metastasis. All of these patents are incorporated by reference herein.
Nothing in these references, however, points to or suggests that galectin-3 has a role in the immunological process known as anergy.
It has now been found that the diminishing or loss of T lymphocyte activity is associated with physical separation of TCR and CD8 molecules. It has also been found that both the proximity of TCR and CD8 and the activity of the T lymphocytes can be restored via treatment of the CD8TLs with appropriate molecules.
How this is accomplished will be seen from the disclosure which follows.